Cloning and expression of a novel galactoside beta 1,3-glucuronyltransferase involved in the biosynthesis of HNK-1 epitope

We isolated a cDNA encoding a novel glucuronyltransferase, designated GlcAT -D, involved in the biosynthesis of the HNK-1 carbohydrate epitope from rat embryo cDNA by the degenerate polymerase chain reaction method. The new cD NA sequence revealed an open reading frame coding for a protein of 324 amin o acids with type II transmembrane protein topology. The amino acid sequenc e of GlcAT-D displayed 50.0% identity to rat GlcAT-P, which is involved in the biosynthesis of the HNK-1 epitope on glycoproteins. Expression of GlcAT -D in COS-7 cells resulted in the formation of the HNK-1 epitope on the cel l surface. The enzyme expressed in COS-7 cells transferred a glucuronic aci d (GlcA) not only to asialo-orosomucoid, a glycoprotein bearing terminal N- acetyllactosamine structure, but also to para-globoside (lacto-N-neotetraos ylceramide), a precursor of the HNK-1 epitope on glycolipids. Furthermore, substrate specificity analysis using a soluble chimeric form of GlcAT-D rev ealed that GlcAT-D transfers a GlcA not only to Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4Glc-pyridylamine but also to Gal beta 1-3GlcNAc beta 1-3Gal bet a 1-4Glc-pyridylamine. Enzymatic hydrolysis and Smith degradation of the re action product indicated that GlcAT-D transfers a GlcA through a beta 1,3-l inkage to a terminal galactose, The GlcAT-D transcripts were detected in em bryonic, post-natal, and adult rat brain. In situ hybridization analysis re vealed that the expression pattern of GlcAT-D transcript in embryo is simil ar to that of GlcAT-P, but distinct expression of GlcAT-D was observed in t he embryonic pallidum and retina. Regions that expressed GlcAT-D and/or Glc AT-P were always HNK-1-positive, indicating that both GlcATs are involved i n the synthesis of the HNK-1 epitope in vivo.